Anti-PECAM Therapy for Metastasis Suppression

ABSTRACT

The invention provides novel compositions, methods, kits, and uses thereof relating to antimetastatic agents useful for treating neoplastic diseases.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/519,986 filed 13 Nov. 2003, which is incorporated by referenceherein.

FIELD OF THE INVENTION

The invention relates to the identification of a method and relatedcompositions for inhibiting the metastatic capability of neoplasticcells in a patient. The methods and compositions comprise aPECAM-binding agent, such as an antibody, and methods of treating orpreventing disease using a PECAM-binding agent to modulatinginvasiveness and metastatic potential of neoplastic cells.

BACKGROUND OF THE INVENTION

Oncogenesis was described by Foulds (1958) as a multistep biologicalprocess, which is presently known to occur by the accumulation ofgenetic damage. On a molecular level, the multistep process oftumorigenesis involves the disruption of both positive and negativeregulatory effectors (Weinberg, 1989). The molecular basis for humancolon carcinomas has been postulated, by Vogelstein and coworkers(1990), to involve a number of oncogenes, tumor suppressor genes andrepair genes. Similarly, defects leading to the development ofretinoblastoma have been linked to another tumor suppressor gene (Lee etal., 1987). Still other oncogenes and tumor suppressors have beenidentified in a variety of other malignancies. Unfortunately, thereremains an inadequate number of treatable cancers, and the effects ofcancer are catastrophic—over half a million deaths per year in theUnited States alone.

Cancer is fundamentally a genetic disease in which damage to cellularDNA leads to disruption of the normal mechanisms that control cellularproliferation. Two of the mechanisms of action by which tumorsuppressors maintain genomic integrity is by cell arrest, therebyallowing for repair of damaged DNA, or removal of the damaged DNA byapoptosis (Ellisen and Haber, 1998; Evan and Littlewood, 1998).Apoptosis, otherwise called “programmed cell death,” is a carefullyregulated network of biochemical events which act as a cellular suicideprogram aimed at removing irreversibly damaged cells. Apoptosis can betriggered in a number of ways including binding of tumor necrosisfactor, DNA damage, withdrawal of growth factors, and antibodycross-linking of Fas receptors. Although several genes have beenidentified that play a role in the apoptotic process, the pathwaysleading to apoptosis have not been fully elucidated. Many investigatorshave attempted to identify novel apoptosis-promoting genes with theobjective that such genes would afford a means to induce apoptosisselectively in neoplastic cells to treat cancer in a patient.

An alternative approach to treating cancer involves the suppression ofangiogenesis with agent such as Endostatin™ or anti-VEGF antibodies. Inthis approach, the objective is to prevent further vascularization ofthe primary tumor and potentially to constrain the size of metastaticlesions to that which can support neoplastic cell survival withoutsubstantial vascular growth.

Platelet endothelial cell adhesion molecule (PECAM-1; CD31) is a proteinfound on endothelial cells and neutrophils and has been shown to beinvolved in the migration of leukocytes across the endothelium. Themodulation of the activity of PECAM-1 for the treatment ofcardiovascular conditions such as thrombosis, vascular occlusion strokeand for the treatment of or for reducing the occurrence of haemostasisdisorders is disclosed in WO03055516A1. PECAM-1 has also been implicatedin the inflammatory process and anti-PECAM-1 monoclonal antibody hasbeen reported to block in vivo neutrophil recruitment (Nakada et al.(2000) J. Immunol. 164: 452-462). PECAM-1 knockout mice have beenreported and appear to have normal leukocyte migration, plateletaggregaton, and vascular development, which implies that there areredundant adhesion molecules which can compensate for a loss of PECAM-1(Duncan et al. (1999) J. Immunol. 162: 3022-3030). Monoclonal antibodiesto PECAM-1 have been reported to block murine endothelial tube formationand related indicators of vascularization in a tumor transplantationmodel (Zhou et al. (1999) Angiogenesis 3: 181-188 and in a human skintransplantation model (Cao et al. (2002) Am. J. Physiol. Cell Physiol.282: C1181-C1190). However, the role of PECAM-1 in tumor angiogenesis,if any, remains undefined.

Despite substantial efforts to inhibit cancer and the metastasis oftumors with anti-angiogenic strategies, to date there are no approvedand marketed drugs for treating cancer solely by the inhibition ofangiogenesis. Indeed the specific roles of various adhesion molecules,including PECAM-1, in the processes of neoplasia and metastasis areunknown.

There exists a need in the art for a method and related compositions forinhibiting the metastatic potential of cancer cells in patients. Thepresent invention fulfills this need and provides related aspectsdesired by practitioners in the field.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention. All patent andliterature publications referenced herein are incorporated by referencefor all purposes as if the entire content of the disclosures weremechanically or electronically reproduced herein.

SUMMARY OF THE INVENTION

The present invention relates to the unexpected discovery that systemicadministration of an antibody that binds to PECAM-1 supresses themetastatic spread of a wide variety of different tumor types which aretypically fatal in humans, and this effect is achieved independent fromany inhibition of angiogenesis, if any. This unexpected discoveryprovides a basis for the generation of novel anticancer treatments andmedicaments, wherein provision of a systemic dosage of anti-PECAMantibody or a proxy that provides the same functional result isadministered to a patient to inhibit or reduce the invasiveness and/ormetastatic potential of neoplastic cells in the patient.

The present invention provides methods for repressing or preventingneoplastic transformation in a cell, the method comprising administeringan anti-PECAM antibody systemically in an amount effective to inhibitthe transformed phenotype and reduce the detectable invasiveness and/ormetastatic potential of the cell. In an embodiment, the anti-PECAMantibody can bind to a PECAM located on a non-neoplastic somatic cell orit can bind to PECAM or a cross-reactive macromolecule present on aneoplastic cell.

An anti-PECAM binding species may be contacted with or introduced to apatient who has been diagnosed with a neoplasm through any of a varietyof manners known to those of skill, however it is often preferred todeliver the anti-PECAM binding agent systemically. With regard to theinvention, an anti-PECAM binding species can comprise an antibody, suchas a humanized or human-sequence monoclonal antibody, and antibodyfragment that comprises F(ab)₂, F(ab′)2, F9ab, F(ab), Dab, Fv, scFv, Fcor a minimal recognition unit of an antibody that has the property ofbinding to human PECAM-1 with an affinity of at least about 1×10⁸ M.Alternative binding species can also include, but are not limited to,proteinaceous binding multimers according to US20030157561A1high-affinity peptides, and equivalents. In some embodiments, theanti-PECAM binding species is covalently linked to poly(ethylene)glycol(PEG), such as a 30K linear PEG, or 40K, 60K, or larger branched PEG—orlarger linear or branched PEG moieties, either via single attachment orvia multiple attachments.

In some embodiments of the present invention, the inventor's discoverythat anti-PECAM binding species administered systemically is able toinhibit metastasis will be used in combination with otheranti-transformation/anti-cancer therapies. These other therapies may beknown at the time of this application, or may become apparent after thedate of this application. For example, a humanized or human sequenceanti-PECAM antibody may be used in combination with other therapeuticpolypeptides, polynucleotides encoding other therapeutic polypeptides,or chemotherapeutic agents. In one representative embodiment, thechemotherapeutic agent is taxol. The anti-PECAM binding species also maybe used in conjunction with radiotherapy. The type of ionizing radiationconstituting the radiotherapy may be selected from the group comprisingx-rays, gamma-rays, and microwaves. In certain embodiments, the ionizingradiation may be delivered by external beam irradiation or byadministration of a radionuclide. The anti-PECAM binding species alsomay be used with gene therapy regimes.

The present invention also provides treatment methods for many humancancers. The treatment method comprises treating a patient having adiagnosed neoplasm, typically a carcinoma or sarcoma or other solidtumor type, with a systemic dosage of anti-PECAM binding speciespreferably delivered via subcutaneous or intravenous administration, orintrathecally into the brain to inhibit brain metastases. Preferredvariations of the method include treating a patient having a diagnosedbreast carcinoma, lung carcinoma, or colon carcinoma by administering aneffective dose of an anti-PECAM binding species, such as a humanized orhuman-sequence anti-PECAM monoclonal antibody via a systemic route suchas subcutaneous or intravenous.

In certain other aspects of the present invention there are providedtherapeutic kits comprising in suitable container, a pharmaceuticalformulation of an anti-PECAM binding species. Such a kit may furthercomprise a pharmaceutical formulation of a therapeutic polypeptide,polynucleotide encoding a therapeutic polypeptide, or chemotherapeuticagent. Such kits may comprise radiosensitizing agents, instructions foradministration of an anti-PECAM binding species to a human patientdiagnosed with a neoplasm—particularly a lung, colon, or breast neoplasmor in a variation a melanoma—via systemic delivery. In a preferredvariation, the kit comprises a humanized or human sequence anti-PECAMmonoclonal antibody which is PEGylated.

The invention also provides antibodies which bind to human PECAM-1 withan affinity of about at least 1×10⁷ M⁻¹ and which lack specific highaffinity binding for a other PECAM-related polypeptides. Such antibodiesmay be used therapeutically by systemic, intracranial, or targeteddelivery to neoplastic cells (e.g., by cationization or by liposome orimmunoliposome delivery).

The invention also provides therapeutic agents which inhibit neoplasia,invasiveness and/or metastasis by modulating PECAM-1 function and whichdo not inhibit angiogenesis; such agents can be used as pharmaceuticals.

The invention provides a method for treating patients who have adiagnosed solid tumor and for whom angiogenesis inhibition would bedetrimental; such as patients having recently suffered a myocardialinfarction, congestive heart failure, stroke, atherosclerosis of thecoronary vessels or cerebrovasculature, or who have a significant woundhealing process resulting from injury or major surgery and which benefitfrom angiogenesis to aid healing or restore circulation.

In a variation of the invention, an immunogenic dose of a denaturedhuman PECAM-1 or a non-human PCAM-1 such as primate, mouse, rat, dog, orpig PECAM-1 protein or a portion thereof is administered to a humanpatient diagnosed with a neoplasm, typically in combination with anadjuvant and/or a covalently-attached or non-attached immunostimulatorypolynucleotide such as those disclosed by Dynavax or ColeyPharmaceuticals. In this way, the human patient is able to make animmune response, including an antibody response, which will crossreactwith their own PECAM-1 protein which they are otherwise tolerized to.

A further understanding of the nature and advantages of the inventionwill become apparent by reference to the remaining portions of thespecification and drawings.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

FIGURES

FIGS. 1A and 1B show the effects of antibody treatment in mice.

FIGS. 2A and 2B show effects of antibody treatment.

FIGS. 3A and 3B show effects of antibody treatment in mice.

FIGS. 4A and 4B show effects of antibody treatment in mice.

FIGS. 5A and 5B show effects of antibody treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The following patent documents are incorporated herein by reference:U.S. Pat. No. 5,968,511; WO0155178; U.S. Pat. No. 6,639,055; U.S. Pat.No. 6,133,426; WO03055516; WO02085405; and U.S. Pat. No.6,627,196—including methods and materials described therein.

Definitions

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. Generally, the term naturally-occurring refers toan object as present in a non-pathological (undiseased) individual, suchas would be typical for the species.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 2, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity.

A “comparison window”, as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 80 percentsequence identity, preferably at least 85 percent identity and often 90to 95 percent sequence identity, more usually at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 nucleotide positions, frequently over a window ofat least 25-50 nucleotides, wherein the percentage of sequence identityis calculated by comparing the reference sequence to the polynucleotidesequence which may include deletions or additions which total 20 percentor less of the reference sequence over the window of comparison. Thereference sequence may be a subset of a larger sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence deduced from a full-length cDNA. Fragments typically are atleast 14 amino acids long, preferably at least 20 amino acids long,usually at least 50 amino acids long or longer, up to the length of afull-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), a biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues. Agents are evaluated for potential activityas antineoplastics, anti-inflammatories, or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which does notsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

The term “protein interaction inhibitor” is used herein to refer to anagent which is identified by one or more screening method(s) of theinvention as an agent which selectively inhibits protein-protein bindingbetween a first interacting polypeptide and a second interactingpolypeptide. Some protein interaction inhibitors may have therapeuticpotential as drugs for human use and/or may serve as commercial reagentsfor laboratory research or bioprocess control. Protein interactioninhibitors which are candidate drugs are then tested further foractivity in assays which are routinely used to predict suitability foruse as human and veterinary drugs, including in vivo administration tonon-human animals and often including administration to human inapproved clinical trials.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a metastasis-prone solid tumortype.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, transcriptional activatorpolypeptide, metal binding domains, epitope tags). In some embodiments,labels are attached by spacer arms of various lengths to reducepotential steric hindrance.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably a substantially purified fraction is a compositionwherein the object species comprises at least about 50 percent (on amolar basis) of all macromolecular species present. Generally, asubstantially pure composition will comprise more than about 80 to 90percent of all macromolecular species present in the composition. Mostpreferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species.

As used herein “normal blood” or “normal human blood” refers to bloodfrom a healthy human individual who does not have an active neoplasticdisease or other disorder of lymphocytic proliferation, or an identifiedpredisposition for developing a neoplastic disease. Similarly, “normalcells”, “normal cellular sample”, “normal tissue”, and “normal lymphnode” refers to the respective sample obtained from a healthy humanindividual who does not have an active neoplastic disease or otherlymphoproliferative disorder.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or nonionic detergents and/or membrane fractionsand/or antifoam agents and/or scintillants.

As used herein, the terms “interacting polypeptide segment” and“interacting polypeptide sequence” refer to a portion of a hybridprotein which can form a specific binding interaction with a portion ofa second hybrid protein under suitable binding conditions. Generally, aportion of the first hybrid protein preferentially binds to a portion ofthe second hybrid protein forming a heterodimer or higher orderheteromultimer comprising the first and second hybrid proteins; thebinding portions of each hybrid protein are termed interactingpolypeptide segments. Generally, interacting polypeptides can formheterodimers with a dissociation constant (K_(D)) of at least about1×10³ M⁻¹, usually at least 1×10⁴ M⁻¹, typically at least 1×10⁵ M⁻¹,preferably at least 1×10⁶ M⁻¹ to 1×10⁷ M⁻¹ or more, under suitablephysiological conditions.

As used herein, the term “multimer” comprises dimer and higher ordercomplexes (trimer, tetramer, pentamer, hexamer, heptamer, octamer,etc.). “Homomultimer” refers to complexes comprised of the same subunitspecies. “Heteromultimer” refers to complexes comprised of more than onesubunit species.

The term “recombinant” used herein refers to PECAM-1 produced byrecombinant DNA techniques wherein the gene coding for protein is clonedby known recombinant DNA technology. For example, the human gene forPECAM-1 may be inserted into a suitable DNA vector, such as a bacterialplasmid, and the plasmid used to transform a suitable host. The gene isthen expressed in the host to produce the recombinant protein. Thetransformed host may be prokaryotic or eukaryotic, including mammalian,yeast, Aspergillus and insect cells.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody fragments solong as they exhibit the desired biological activity.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (VH) followed by a number of constant domains.Each light chain has a variable domain at one end (VL) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR) regions. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institute of Health, Bethesda,Md. [1991]) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Clothia and Lesk, J. Mol. Biol., 196:901-917[1987]). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′,F(ab′).sub.2, and Fv fragments; diabodies; linear antibodies (Zapata etal., Protein Eng., 8(10): 1057-1062 [1995]); single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′).sub.2 fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (.kappa.) and lambda (.lambda.), based on the amino acid sequencesof their constant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are known.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, ie.,the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256:495 [1975], or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. For further details, see, Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988]; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody include PRIMATIZED™ antibody wherein the antigen-binding regionof the antibody is derived from an antibody produced by immunizingmacaque monkeys with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V.sub.H andV.sub.L domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V.sub.H and V.sub.L domainswhich enables the sFv to form the desired structure for antigen binding.For a review of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V.sub.H) connected to a light-chain variable domain (V.sub.L) inthe same polypeptide chain (V.sub.H-V.sub.L). By using a linker that istoo short to allow pairing between the two domains on the same chain,the domains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

DETAILED DESCRIPTION OF THE INVENTION

The nomenclature used hereafter and the laboratory procedures in cellculture, molecular genetics, and nucleic acid chemistry andhybridization described below may involve well known and commonlyemployed procedures in the art. Standard techniques are used forrecombinant nucleic acid methods, polynucleotide synthesis, andmicrobial culture and transformation (e.g., electroporation,lipofection). The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see, generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference)which are provided throughout this document.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Applications for DNA Amplification ed. H A Erlich,Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide to Methodsand Applications, eds. Innis, Gelfland, Snisky, and White, AcademicPress, San Diego, Calif. (1990); Mattila et al. (1991) Nucleic AcidsRes. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

Production and Applications of α-PECAM Antibodies

Native human PECAM-1 proteins, fragments thereof, or analogs thereof,may be used to immunize an animal for the production of specificantibodies. These antibodies may comprise a polyclonal antiserum or maycomprise a monoclonal antibody produced by hybridoma cells. For generalmethods to prepare antibodies, see Antibodies: A Laboratory Manual,(1988) E. Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., which is incorporated herein by reference.

For example but not for limitation, a recombinantly produced fragment ofPECAM-1 can be injected into a mouse along with an adjuvant followingimmunization protocols known to those of skill in the art so as togenerate an immune response. Typically, approximately at least 1-50 μgof a PECAM-1 fragment or analog is used for the initial immunization,depending upon the length of the polypeptide. Alternatively or incombination with a recombinantly produced PECAM-1 polypeptide, achemically synthesized peptide having a PECAM-1 sequence may be used asan immunogen to raise antibodies which bind a PECAM-1 protein, such asthe native PECAM-1 polypeptide having the sequence shown essentially inFIG. 1( a), a native human PECAM-1 polypeptide, a polypeptide comprisinga PECAM-1 epitope, or a PECAM-1 fusion protein. Immunoglobulins whichbind the recombinant fragment with a binding affinity of at least 1×10⁷M⁻¹ can be harvested from the immunized animal as an antiserum, and maybe further purified by immunoaffinity chromatography or other means.Additionally, spleen cells are harvested from the immunized animal(typically rat or mouse) and fused to myeloma cells to produce a bank ofantibody-secreting hybridoma cells. The bank of hybridomas can bescreened for clones that secrete immunoglobulins which bind therecombinantly-produced PECAM-1 polypeptide (or chemically synthesizedPECAM-1 polypeptide) with an affinity of at least 1×10⁶ M⁻¹. Animalsother than mice and rats may be used to raise antibodies; for example,goats, rabbits, sheep, and chickens may also be employed to raiseantibodies reactive with a PECAM-1 protein. Transgenic mice having thecapacity to produce substantially human antibodies also may be immunizedand used for a source of α-PECAM-1 antiserum and/or for makingmonoclonal-secreting hybridomas.

Bacteriophage antibody display libraries may also be screened forbinding to a PECAM-1 polypeptide, such as a full-length PECAM-1 protein,a PECAM-1 fragment, or a fusion protein comprising a PECAM-1 polypeptidesequence comprising a PECAM-1 epitope (generally at least 3-5 contiguousamino acids). Generally such PECAM-1 peptides and the fusion proteinportions consisting of PECAM-1 sequences for screening antibodylibraries comprise about at least 3 to 5 contiguous amino acids ofPECAM-1, frequently at least 7 contiguous amino acids of PECAM-1,usually comprise at least 10 contiguous amino acids of PECAM-1, and mostusually comprise a PECAM-1 sequence of at least 14 contiguous aminoacids.

Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, a bacteriophageantibody display library is screened with a PECAM-1 polypeptide that isimmobilized (e.g., by covalent linkage to a chromatography resin toenrich for reactive phage by affinity chromatography) and/or labeled(e.g., to screen plaque or colony lifts).

PECAM-1 polypeptides which are useful as immunogens, for diagnosticdetection of α-PECAM-1 antibodies in a sample, for diagnostic detectionand quantitation of PECAM-1 protein in a sample (e.g., by standardizedcompetitive ELISA), or for screening a bacteriophage antibody displaylibrary, are suitably obtained in substantially pure form, that is,typically about 50 percent (w/w) or more purity, substantially free ofinterfering proteins and contaminants. Preferably, these polypeptidesare isolated or synthesized in a purity of at least 80 percent (w/w)and, more preferably, in at least about 95 percent (w/w) purity, beingsubstantially free of other proteins of humans, mice, or othercontaminants.

For some applications of these antibodies, such as identifyingimmunocrossreactive proteins, the desired antiserum or monoclonalantibody(ies) is/are not monospecific. In these instances, it may bepreferable to use a synthetic or recombinant fragment of PECAM-1 as anantigen rather than using the entire native protein. Production ofrecombinant or synthetic fragments having such defined amino- andcarboxy-termini is provided by the PECAM-1.

If an antiserum is raised to a PECAM-1 fusion polypeptide, such as afusion protein comprising a PECAM-1 immunogenic epitope fused toβ-galactosidase or glutathione S-transferase, the antiserum ispreferably preadsorbed with the non-PECAM-1 fusion partner (e.g,β-galactosidase or glutathione S-transferase) to deplete the antiserumof antibodies that react (i.e., specifically bind to) the non-PECAM-1portion of the fusion protein that serves as the immunogen. Monoclonalor polyclonal antibodies which bind to the human and/or murine PECAM-1protein can be used to detect the presence of human or murine PECAM-1polypeptides in a sample, such as a Western blot of denatured protein(e.g., a nitrocellulose blot of an SDS-PAGE) obtained from a lymphocytesample of a patient. Preferably quantitative detection is performed,such as by denistometric scanning and signal integration of a Westernblot. The monoclonal or polyclonal antibodies will bind to the denaturedPECAM-1 epitopes and may be identified visually or by other opticalmeans with a labeled second antibody or labeled Staphylococcus aureusprotein A by methods known in the art.

One use of such antibodies is to screen cDNA expression libraries,preferably containing cDNA derived from human or murine mRNA fromvarious tissues, for identifying clones containing cDNA inserts whichencode structurally-related, immunocrossreactive proteins, that arecandidate novel PECAM-1 binding factors or PECAM-1-related proteins.Such screening of cDNA expression libraries is well known in the art,and is further described in Young et al., Proc. Natl. Acad. Sci. U.S.A.80:1194-1198 (1983), which is incorporated herein by reference) as wellas other published sources. Another use of such antibodies is toidentify and/or purify immunocrossreactive proteins that arestructurally or evolutionarily related to the native PECAM-1 protein orto the corresponding PECAM-1 fragment (e.g., functional domain;PECAM-1-interacting protein binding domain) used to generate theantibody. The anti-PECAM-1 antibodies of the invention can be used tomeasure levels of PECAM-1 protein in a cell or cell population, forexample in a cell explant (e.g., lymphocyte sample) obtained from apatient. The anti-PECAM-1 antibodies can be used to measure thecorresponding protein levels by various methods, including but notlimited to: (1) standardized ELISA on cell extracts, (2)immunoprecipitation of cell extracts followed by polyacrylamide gelelectrophoresis of the immunoprecipitated products and quantitativedetection of the band(s) corresponding to PECAM-1, and (3) in situdetection by immunohistochemical straining with the anti-PECAM-1antibodies and detection with a labeled second antibody. The measurementof the ratio of PECAM-1 to control housekeeping proteins in a cell orcell population is informative regarding the invasive and metastaticstatus of the cell or cell population.

An antiserum which can be utilized for this purpose can be obtained byconventional procedures. One exemplary procedure involves theimmunization of a mammal, such as rabbits, which induces the formationof polyclonal antibodies against PECAM-1. Monoclonal antibodies are alsobeing generated from already immunized hamsters. This antibody can beused to detect the presence and level of the PECAM-1 protein.

It is also possible to use the proteins for the immunological detectionof PECAM-1 and associations thereof with standard assays as well asassays using markers, which are radioimmunoassays or enzymeimmunoassays.

The detection and determination of PECAM-1 has significant diagnosticimportance. For example, the detection of a PECAM-1 decline favoringinvasiveness and metastasis would be advantageous in cancer therapy andcontrolling hypertrophies. The detection or determination of proteinsfavoring metastasis and invasion will be beneficial in detecting anddiagnosing cancer, neurodegenerative diseases, and ischemic cell death.Thus these proteins and their antibodies can be employed as a marker tomonitor, check or detect the course of disease.

Cross-linked complexes of PECAM-1 with PECAM-1-interacting polypeptidescan be used as immunogens, and the resultant antisera preadsorbed withPECAM-1 and PECAM-1-interacting polypeptide such that the remainingantisera comprises antibodies which bind conformational epitopes presenton the complexes but not the monomers (e.g., complex-specific epitopes).Complex-specific hybridomas and monoclonal antibodies can be similarlygenerated. Such antibodies can be used diagnostically to detect andquantitate the presence of specific complexes and correlate this datawith disease or cell type, and the like.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)].The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human monoclonal antibodies (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) andBoerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, humanantibodies can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859(1994); Morrison, Nature, 368:812-13 (1994); Fishwild et al., NatureBiotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826(1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed.[1980]), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The anti-PECAM binding species of the present invention can beadministered to a cancer patient in conjunction with otherchemotherapeutic agents and radiotherapy sensitizers.

The following examples are given to illustrate the invention, but arenot to be limiting thereof. All percentages given throughout thespecification are based upon weight unless otherwise indicated. Allprotein molecular weights are based on mean average molecular weightsunless otherwise indicated.

The foregoing description of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in light of the above teaching.

Such modifications and variations which may be apparent to a personskilled in the art are intended to be within the scope of thisinvention.

EXPERIMENTAL EXAMPLES Materials and Methods

Female mice 6-8 weeks of age were used for all studies. C57B1/6 andBalbC mice were purchased from Simonson Labs, (Gilroy, Calif.), and theNu/Nu mice were purchased from Charles River. Tumor cells wereinoculated by tail vein injection. For the B16-F10 murine melanoma tumorand Lewis Lung carcinoma highly metastatic (LLC-HM) lung cancer models,each C57B1/6 mouse received 25,000 tumor cells suspended in 200 μl ofculture media. For the 4T1 murine breast cancer and CT26 murine coloncancer models, each Balb/C mouse received 50,000 tumor cells tumor cellssuspended in 200 μl of culture media. For the Lox cell human melanomaxenograft model, Nu/Nu mice received a total of 2.5 million Lox cells inculture media in two separate tail vein injections. The first injectionwas administered in the morning and the second injection four hourslater. Each injection contained 1.25 million cells in 300 μl of culturemedia.

For the first set of experiments, groups of eight mice received 25,000B16-F10 tumor cells by tail vein injection on day 0, and then received 5doses of 200 μg of either rat anti-mouse anti-PECAM-1 (mAb 390)(provided by Dr. H. Delisser, University of Pennsylvania) or Rat IgG2(a)isotype control antibody (Sigma) by the following schedules. One groupof mice received 5 doses of 200 μg of either rat anti-mouse anti-PECAM-1(mAb 390) or Rat IgG2(a) isotype control antibody starting on day 0(just after tumor cell injection), and then on days 1, 3, 6 and 8. Inaddition, one group of mice received 5 doses of 200 μg of either ratanti-mouse anti-PECAM-1 (mAb 390) or Rat IgG2(a) isotype controlantibody starting on day 7 after tumor cell injection, and then on days8, 10, 13 and 15. Mice bearing CT26, 4T1 B16 and Lox cells weresacrificed on days 20, 22, 23, and 28 after tumor cell inoculation,respectively.

In each case, all mice from the respective group were sacrificed when anindex animal looked seriously ill or died, and significant numbers oflung tumors were documented following sacrifice and analysis ofdissected lungs. Lungs from each mouse were dissected out and thenweighed. The lungs then were infused intra-tracheally with 5% bufferedformalin for mice bearing B16-F10 melanoma. For all other tumor-bearingmice (the 4T1, CT26, LLC-HM and Lox tumor models), the lungs were thenwere infused intra-tracheally with the fixative solution containingIndian ink. All lung samples were then fixed in 5% buffered formalin(50% of 10% buffered formalin (Fisher) and 50% PBS). Lung tumors werecounted under a dissecting microscope by an observer blinded to whichgroup from which they came. The potential significance of differencesbetween various groups was assessed using an unpaired, two-sidedStudent's t Test.

Subsequently, the lungs were subjected to the following studies. Mitoticand apoptotic figures were counted on four-micron hematoxylin and eosinstained slides using a conventional light microscope. Actual counts ofmitotic and apoptotic figures were made from the ten largest nodules.Apoptotic bodies and mitotic figures were counted according topreviously described morphologic criteria (1, 2). The apoptotic andmitotic rate were calculated based on the degree of tumor cellularity,and expressed as the number of apoptotic or mitotic figures per thousandcells. In situ detection of cleaved, apoptotic DNA fragments (TUNEL) wasperformed using the TdT-FragEL Detection Kit (Oncogene Science)according to the manufacturer's protocol. The frequency of labeled cellswas calculated by counting at least 1,000 cells in areas with thehighest number of TdT labeled nuclei. Matrigel assay and Boyden chamberanalysis were performed as described (3). All analyses of apoptosis,mitosis, angiogenesis and histopathology were performed by aninvestigator blinded to the identity of the specimens being assessed.Expression of PECAM-1 on the various tumor cell lines assessed wasperformed by FACS analysis.

REFERENCES

-   1. Kerr, J. F., Wyllie, A. H., & Currie, A. R. (1972) Br. J. Cancer    26, 239-257.-   2. van Diest, P. J., Brugal, G., & Baak, J. P. (1998) J. Clin.    Pathol. 51, 716-724.-   3. Desprez, P. Y., Lin, C. Q., Thomasset, N., Sympson, C. J.,    Bissell, M. J., & Campisi, J. (1998) Mol. Cell. Biol. 18, 4577-4588.

Results of the Anti-Metastatic Tumor Studies:

Binding of anti of anti-PECAM-1 antibody to the various tumor cell linestested.

Binding of anti-PECAM-1 antibody to murine B16-F10 melanoma cells,murine 4T1 mammary carcinoma cells and murine Lewis Lungcarcinoma-highly metastatic (LLC-HM) cells was assessed.

No PECAM-1 expression (no specific binding of anti-PECAM-1 antibody toany of these cell types was detected (data not shown)). Analysis ofPECAM-1 expression on murine CT26 colon tumor cells and human LOXmelanoma cells is pending.

Effects of anti-PECAM-1 on the metastatic progression of B16-F10melanoma. We first compared the potential anti-tumor effects of five,200 μg intravenous doses of either anti-PECAM-1 or IgG isotype controlantibody, with treatment initiated either on the day of tumor injection(day 0) or 7 days after tumor cell injection (day 7). Injection of IgGisotype control antibody, beginning on either day 0 or day 7, as well asinjection of anti-PECAM-1 antibody beginning on day 0 had no effect oneither total lung weight (an indicator of overall metastatic burden) orthe total number of metastatic lung tumors, when compared to untreated,tumor-bearing control mice. The lack of anti-tumor efficacy produced byanti-PECAM antibody therapy initiated on day 0 contrasts with theresults of a prior study testing this dose and schedule of anti-PECAMantibody against locally-inoculated, sub-cutaneous B16 melanoma tumors.In this prior study, five, 200 μg intraperitoneal doses of the sameanti-PECAM-1 antibody begun on the same day (day 0) as localsub-cutaneous inoculation of B16 melanoma tumors did produce significantanti-tumor activity, significantly reducing both local tumor growth, aswell as significantly reducing tumor angiogenesis (Zhou et al.Angiogenesis 3: 181-188, 1999). In contrast, our studies demonstratedthat injection of this same anti-PECAM-1 antibody beginning on day 0showed no anti-tumor activity against B16 melanoma lung metastases.However, we discovered that intravenous injection of anti-PECAM-1antibody beginning on day 7 after tumor cell injection was highlyeffective against metastatic B16 melanoma tumors, significantly reducingboth total lung weights (p 0.005) and the total number of metastaticB16-F10 melanoma lung tumors (p<0.0001), when compared to control mice(FIGS. 1A and 1B). Thus, anti-tumor results obtained usingIP-administered anti-PECAM antibody against local tumors can differsubstantially from those obtained using intravenously-injectedanti-PECAM antibody against metastatic tumors.

We then attempted to repeat these results in a follow-up experiment,again comparing the effects of either the anti-PECAM-1 antibody or theisotype control, initiated 7 days after IV injection of B16-F10 cells.We found that anti-PECAM-1 antibody significantly reduced both totallung weights (p<0.05) and the total number of lung tumors (p<0.0001)when compared to B16-F10-bearing mice treated with the same schedule anddose of isotype control antibody (FIGS. 2A and 2B). (We used isotypecontrol antibody treated mice as controls in all subsequent experimentsbecause we previously showed that total lung weights and total numbersof lung tumors do not differ between mice treated with isotype controlantibody and untreated mice (see FIGS. 1A and 1B)). Anti-PECAM antibodytherapy initiated on day 7 after tumor cell injection againsignificantly reduced overall tumor burden and the total number of lungmetastatic tumors, as we had previously observed in experiment 1 above.Intraperitoneal administration of this same anti-PECAM antibody haspreviously been reported to significantly reduce tumor angiogenesis insubcutaneously inoculated B16 melanoma tumors. We assessed tumorangiogenesis, as well tumor apoptotic and mitotic rates in B16-F10 lungtumors from the anti-PECAM- and isotype control antibody-injectedgroups. Surprisingly, the number of blood vessels in lung tumorsappeared higher in the anti-PECAM-treated group (17.9+4.5 tumor bloodvessels/HPF (avg+S.E.)) than the isotype control-treated group(8.79+2.9), although this difference did not approach statisticalsignificance (p=0.12). The level of tumor apoptosis (9.9+1.1) inanti-PECAM- versus isotype control-treated mice (9.7+0.8) was alsocomparable. However, the rate of mitosis in tumor cells wassignificantly higher (p<0.05) in isotype control-treated mice (4.7+0.5)versus anti-PECAM-treated mice (2.9+0.7). Histopathologically, tumornecrosis, hemorrhage, pulmonary congestion and/or intravascular emboliwere noted in 7 of 9 isotype control antibody-treated mice, whereas noneof these findings were noted in anti-PECAM-1 antibody-treated mice (datanot shown). Overall, anti-PECAM antibody therapy significantly reducedthe total numbers of metastatic B16-F10 tumors and overall tumor burden,as well as significantly reducing tumor cell mitotic rates and lunghistopathologic changes. Anti-PECAM-1 antibody therapy did not reduceeither tumor angiogenesis or tumor apoptosis.

To demonstrate that the anti-metastatic activity of anti-PECAM-1antibody was specific for a broad spectrum of solid tumors, in additionto B16-F10 melanoma tumors, we then tested its potential anti-metastaticactivity against a variety of other tumor cell lines injected into mice.These lines included murine 4T1 mammary carcinoma cells, murine CT26colon tumor cells, murine Lewis Lung carcinoma-highly metastatic(LLC-HM) cells and human LOX melanoma cells. We used establishedprotocols for generating the metastatic spread of each of these lines,as described in the materials and methods section above. We found thatfive, 200 μg doses of IV, anti-PECAM antibody therapy initiated on day 7produced significant anti-metastatic activity against each of thesetumor lines in tumor-bearing mice.

Specifically, anti-PECAM antibody was highly effective against themetastatic spread of 4T1 mammary carcinoma tumors, producing significantreductions of total tumor burden (p<0.005) and total metastatic lungtumors (p<0.0001), when compared to isotype control antibody-treatedmice (see FIGS. 3A and 3B). Again contrary to previous reports byothers, even though anti-PECAM antibody therapy was highly effectiveagainst the metastatic spread of 4T1 tumors, it had no effect on tumorvascularity, since anti-PECAM-treated mice showed 27.8+2.5 tumor bloodvessels/HPF, whereas isotype control-treated mice showed 26.5+2.1 tumorblood vessels/HPF (data not shown). Thus, unlike the anti-tumor effectsof this same anti-PECAM antibody against sub-cutaneous tumors (Zhou etal. Angiogenesis 3: 181-188, 1999), anti-PECAM antibody effects againstmetastatic tumors does not appear to be mediated through effects ontumor angiogenesis. (The analysis the effects of anti-PECAM antibody ontumor mitotic and apoptotic rates for 4T1 tumors is in progress).

Anti-PECAM antibody was less active against the metastatic spread ofCT26 colon tumors, but still significantly reduced the total number ofmetastatic lung tumors (p<0.05), when compared to isotype controlantibody-treated mice (see FIGS. 4A and 4B).

Anti-PECAM antibody therapy did not significantly reduce (p=0.74) lungweights in mice bearing LLC-HM tumors (0.85±0.2 gm) when compared toisotype control antibody-treated mice (0.94±0.1 gm). Since lungmetastases grew largely as confluent masses rather than discrete tumorsin this experiment, it was not possible to accurately count the numbersof individual lung tumors in mice. However, the number of extrapulmonarylung metastases appeared to be significantly reduced in the anti-PECAMantibody treated mice. Specifically, all eight of eight isotypecontrol-treated mice showed discrete, bulky extrapulmonary tumors in thethoracic cavity, whereas of only 3 of 9 anti-PECAM antibody treated miceshowed extrapulmonary tumors. (Note, one of the nine isotype controlantibody-treated mice died with an extensive tumor burden 4 days beforeall other mice were sacrificed. This mouse was not included in the finalanalysis of metastatic LLC-HM tumors. In addition, 3 of 8 isotypecontrol-treated mice showed liver metastases, whereas of only 1 of 9anti-PECAM antibody treated mice showed liver tumors. Thus, while notclearly reducing the number of lung metastatic LLC-HM tumors, anti-PECAMantibody therapy did appear to significantly reduce the extrapulmonaryspread of LLC-HM tumors.

Last, since anti-PECAM antibody therapy reduced the metastatic spread offour different murine solid tumors in immunocompetent, syngeneic mousestrains, we assessed whether anti-PECAM antibody therapy altered themetastatic spread of human LOX xenograft tumors in nude mice. As in themurine tumor models, anti-PECAM antibody therapy significantly reducedthe metastatic spread of LOX cells in nude mice, as measured by bothlungs weights (p<0.05) and the total number of lung tumors (p<0.005)when compared to LOX-bearing mice treated with isotype control antibody(see FIGS. 5A and 5B).

Taken together, these data show that systemic, anti-PECAM antibodytherapy can significantly reduce the metastatic spread of a wide varietyof common fatal solid tumors (melanoma, breast, colon and lung cancer)in mouse metastasis models. Importantly, none of the tumor cells testedexpress detectable PECAM-1, indicating that anti-PECAM antibody does notproduce its anti-metastatic tumor effects via direct binding to thetumor cells themselves. Rather, anti-PECAM antibody appears to functionas an anti-tumor agent via binding to PECAM-1 expressed on vascularendothelial cells. However, anti-PECAM antibody does not appear exertanti-metastatic activity by effects on tumor blood vessel formation.Thus, unlike anti-tumor antibodies currently used to treat humancancers, anti-PECAM is neither tumor type specific, nor does it requireexpression of its cognate receptor on tumor cells to produce significantanti-metastatic effects.

1-13. (canceled)
 14. A kit for repressing or preventing metastasis orinvasiveness of a neoplastic cell in a mammal, the kit comprising atherapeutically effective amount of an anti-PECAM binding species andinstructions for systemic administration of said anti-PECAM bindingspecies to said mammal.
 15. The kit of claim 14, wherein the anti-PECAMbinding species is a monoclonal antibody.
 16. The kit of claim 15,wherein the instructions specify administration of a plurality of doseswherein each dose comprises at least 200 micrograms of said monoclonalantibody.
 17. The kit of claim 16, wherein the instructions specify thatsaid monoclonal antibody is administered in at least five doses witheach dose separated by at least two days from other doses in theplurality.
 18. The kit of claim 15, wherein the instructions specifythat the monoclonal antibody may be combined with other anti-neoplastictherapies.
 19. The kit of claim 18, wherein the other anti-neoplastictherapies comprise: ionizing radiation, chemotherapeutic agents, thermalabalation, anti-tumor monoclonal antibodies that do not bind to PECAM,implantation of radioisotopes seeds, and surgery.
 20. The use of ananti-PECAM monoclonal antibody to repress or prevent metastasis of aneoplastic cell in a mammal wherein said mammal has had neoplastic cellsin its body for at least seven days prior to administration of saidanti-PECAM monoclonal antibody.